PROJECT SUMMARY/ABSTRACT Few molecules in human cells contain esters. Likewise, human cells have high esterase activity. That activity is exploited routinely for the delivery of pharmacological agents as prodrugs in which carboxyl moieties are masked as esters. The esters in prodrugs are typically made with ethanol, which is not toxic as a byproduct of ester hydrolysis. Although esterases vary in different tissues and cell types, variations in esterase specificity are not exploited in the current pharmacopeia. The proposed research seeks to provide the technological basis for tailoring the alcohol portion of prodrugs (and latent probes) to particular physiological contexts. Work will begin by developing a high-throughput assay for the esterase-catalyzed hydrolysis of alkyl esters in which the alcohol protein of the ester is a variable. Then, a library of esters will be synthesized by reacting alcohols with a common intermediate. Finally, a profile of esterase substrate specificity will be developed across a wide range of tissues, cell types, and sera. The ensuing profile of the substrate specificity of esterases endogenous in contexts most relevant for both drug development and basic research in chemical biology will enable future efforts to take prodrug and latent probe design into a new realm.